4Department of Developmental and Molecular Biology,

Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite that is important human and animal pathogen. Experimental information on T. gondii membrane proteins is limited, and the majority of gene predictions with predicted transmembrane motifs are of unknown function. A systematic analysis of the membrane proteome of T. gondii is important not only for understanding this parasite’s invasion mechanism(s), but also for the discovery of potential drug targets and new preventative and therapeutic strategies. Here we report a comprehensive analysis of the membrane proteome of T. gondii, employing three proteomics strategies: 1D gel LC-MS/MS analysis (one-dimensional gel electrophoresis liquid chromatography-tandem mass spectrometry), biotin labeling in conjunction with 1D gel LC-MS/MS analysis, and a novel strategy that combines three-layer “Sandwich ” Gel Electrophoresis (TLSGE) with multidimensional protein identification technology (MudPIT). A total of 2,241 T. gondii proteins with at least one predicted transmembrane segment were identified and grouped into 84